15.6.79
Specialia
ciation with this species. Examination at X500 magnification also failed to reveal the presence of obvious contaminant microorganisms. Homogenization of 300 g of freshly collected L birotulata with 600 ml of 0.1 M, pH 7.4 potassium phosphate buffer, followed by centrifugation for 20 rain at 11,000 rpm and recentrifugation of the supernatant for 30 rain at 15,000 rpm (DuPont Sorvall SS-34) gave a dark purple-colored supernatant. The protein precipitate resulting from the 2550% (NH4)2SO 4 fractionation of this supernatant was resuspended in the original volume of phosphate buffer to give the enzyme preparation of interest. Standard assays for peroxidase employing pyrogallol 1~ and dianisidinel~were used to monitor this partial purifcation. The enzyme preparation at this point was only a 2-fold purified product with respect to total protein, but was almost free of the contaminating purple pigment; thus, enabling us to employ spectrophotometric methods in studies of its peroxidase activities. Results and discussion. The oxidation of pyrogallol by the enzyme preparation occurred only in the presence of H202, and was inhibited by both KCN and NaN 3 at 0.01% concentrations. Although the enzyme retained most of its activity upon standing for several days at room temperature, gentle heating destroyed the activity. The initial rate of oxidation and total amount of pyrogallol oxidized within 5 rain was directly related to the amount of enzyme preparation employed (figure). The peroxidase activity was observed throughout the pH range of 4.0-8.0, with 7.5 being the optimal pH. Similar results were obtained with dianisidine as the substrate, although solubility problems prevented pH dependency studies above pH 6.0. The enzyme preparation was unable to effect chlorination of monochlorodimedon a0, nor did it convert 4-chloroaniline to 4-chloronitrosobenzene 6. Thus, we conclude that the peroxidase in L birotulata is similar in properties to horseradish peroxidase rather than chloroperoxidase.
719
Based on pyrogallol oxidation at pH 7.5, the level of peroxidase activity is estimated to be between 12 and 15 ~tM units per g of fresh sponge tissue. This represents an order of magnitude greater concentration of peroxidase in this sponge when compared to a marine red alga known to possess a high level of peroxidase 12. This surprisingly high level of peroxidase activity that we observed in L birotulata makes it highly improbable that this activity could be due to microalgae that might be present on the sponge surface. Our demonstration of the presence of a peroxidase in this sponge necessitates a reconsideration of this biochemical parameter in the Porifera. A survey of sponges for peroxidative enzymes is now underway with the hopes of developing a potential chemotaxonomic tool for the classification of Porifera.
1 This study was supported by grant No.CA 21992 from the National Cancer Institute, DHEW. 2 L. Minale, G. Cimino, S. de Stefano and G. Sodano, Fortschr. Chem. org. NatStoffe 33, 1 (1976). 3 R.J. Wells, Tetrahedron Lett. 2637 (1976). 4 W. Fenical, J. Phycol. 11, 245 (1975). 5 K.H. Wurzinger and R. Hartenstein, Comp. Biochem. Physiol. 49B, 171 (1974). 6 M.D. Corbett, B.R. Chipko and D.G. Baden, Biochem. J. 175, 353 (1978). 7 M.D. Corbett, B.R. Chipko and J.H. Paul, J. envir. Path. Tox. 1, 259 (1978). 8 G.L. Voss, "Seashore Life of Florida and the Caribbean," E.A. Seemann Co., Miami, 1976, p.26. 9 F. Wiedenmayer, Shallow Water Sponges of the Western Bahamas, p. 138. Birkh~user Verlag, Basel 1977. 10 P.F. Hollenberg, T. Rand-Meir and L.P. Hager, J. biol. Chem. 249, 5816 (1974). 11 R. Bartha and L. Bordeleau, Soil Biol. Biochem. 1, 139 (1969). 12 M.J. Murphy and C. Oh Eocha, Phytochemistry 12, 55 (1973).
T h e glutathione status of the rat liver * G. Harisch, J. Eikemeyer and J. Schole
Institute of Physiological Chemistry, College of Veterinary Medicine Hannover, Bisehofsholer Damm 15, D-3000 Hannover (Federal Republic of Germany), 16 October 1978 Summary. F o r the determination of cellular total glutathione, a new method is presented based on a fluorometric procedure. The relation between reduced glutathione, mixed glutathione disulfides and disulfide glutathione will be designated the glutathione status. The tripeptide glutathione is a structural component of most living cells 1. It can be found in 3 forms, as reduced glutathione (GSH), as symmetrical glutathione disulfide (GSSG), and as unsymmetrical glutathione disulfide which we will call XSSG z. In the last form, the glutathione has combined with cysteine-SH groups of cellular proteins ~-9 or with low molecular SH compounds of the cell 1~ to form a disulfide bond. By this means, a 'depot' is developed which, however, in certain situations can very rapidly be mobilized and then is available to the cell as GSH 11. This characteristic makes it appear desirable to determine, in all investigations of cellular thiol-disulfide-status, the percent glutathione bound but mobilizable. Normally this would be done by reducing the disulfide bonds and then measuring the increased G S H level.
However, as there are often only very small quantities available in biological studies, a sensitive and selective GSH assay is necessary. A fluorometric method with these prerequisites was recently presented by Hissin and Hilf 12. We have adapted this method to the conditions in liver tissues and expanded it to include the cellular total glutathione (TG), and thus the portion of bound glutathione can be measured as well. Materials and methods. Male SPF rats, Wistar Han/B6 were kept under defined conditions (24 ~ light from 06.00 till 18.00 h) until they reached the desired 120 g. The animals were anesthetized with 40 mg/kg nembutal i.p. (Pentobarbital Sodium Abbott). The left lobe of the liver was removed using the freeze stop method 13,14. T G measurement: The measurement of cellular total gluta-
720
Specialia
Experientia 35/6
Table 1. Total glutathione (TG) and free glutathione (GSH) GSH added (gg per 80 mg liver) 0 45 TG (gg per 80 mg liver) Expected value Recovery (%) GSH (pg per 80 mg liver) Expected value Recovery (%)
195 + 6.4
139_+5.0
238 _+2.9 240 99 174_+ 1.2 184 95
90
113
135
284-+ 4.4 285 99 226_+ 3.4 229 99
297 -+9.3 308 96 250+_0.4 252 99
326-+ 5.9 330 98 262_+2.4 274 96
Means___SD of 14 liver homogenates from 14 male Wistar rats (120 g b. wt); GSH was added before the homogenation.
thione was done according to the method proposed by Habeeb for splitting smaller amounts of disulfides with NaBH415; however, this method was modified for the conditions necessary for fluorometric G S H measurement: 80 m g fiver powder in 1.75 ml Tris buffer (0.1 M, p H 8.0), in which immediately before the experiment 100 mg NaBH 4 was dissolved, and 2.0 ml guanidine (8 M) were homogenized for 20 sec in an Ultra-Turrax (Jahnke and Kunkel, Staufen, Germany); 0.05 ml n-octanol was used to prevent foaming. The homogenate was incubated (40 ~ for 30 m i n and then cooled in an icebath. 1.0 ml 50% ice cold (HPO3) . was used to deproteinize the sample, which was then centrifuged for 15 rain at 104,000 x g in a vertical rotor (TV 865, D u Pont-Sorvall). 0.5 ml of the supernatant was diluted with 4.5 ml phosphate EDTA-buffer (0.1 M potassium phosphate, 0.005 M EDTA, p H 8.0) T h e n 0.1 ml of the above dilution, 1.8 ml phosphate E D T A buffer and 0.1 ml o-phthalaldehyde (OPT) solution (100 Ixg OPT) were combined and mixed well. After 15 m i n at room temperature, the mixture was transferred to a fluorescence cuvette. The fluorescence intensity was measured in a Shimadzu R F 500 at 423 n m (activation at 350 nm). GSH measurement: The G S H m e a s u r e m e n t corresponded to that of Hissin and Hilf lz. G S H values obtained by the fluorometric method coincide with those determined by the glyoxalase method 1,z. Other low molecular thiols reacting with OPT are quantitativelynegligible 12. G S S G measurement: As the proposed method from Hissin and t-Iilf for measuring fluorometric G S S G 12 is not reliable 16, disulfide glutathione was measured using the enzymatic optic method with glutathione reductase and N A D P H 17. Results and discussion. Glutathione parameters show diurnal 18 and probably also a n n u a l rhythms, and they are dependent on body weight 2. Data o n glutathione in the literature are hardly comparable as they were seldom obtained under identical conditions. 1. Data on T G (table 1) are rather sparse in the literature 3'6. The only exception is the lens of the h u m a n eye 19. Reduced glutathione (GSH) (table 1) is well investigated. Our data are in good agreement with the liver GSH-values obtained with other methods 12'2~ 2. XSSG is calculated by the
Table 2. Glutathione status TG (as GSH)
GSH
gg per g liver 2 4 4 5 _ + 8 0 1741+62 ixmoles per g liver 7.96 5.67 Number of animals 14 14
XSSG ~ (as GSH)
GSSG
636 2.07
67+4 0.110 14
The values are means+_SD; male Wistar rats with 120 g b.wt; see 'results and discussion' section.
formula X S S G = T G - ( G S H + 2 GSSG); XSSG is expressed as SH-glutathione and all data are given in ~tmoles per g liver (table 2). Our results are in coincidence with those of Harrap 3 which were obtained with a different method, and they are in agreement with the results found by Modig with aszites t u m o u r cells 6. 3. The same GSSG-values were obtained by Tietze 2~with a different method. Some authors try to prevent possible G S H oxidation occuring during tissue preparation and they report lower GSSG-values 24,25. 4. The relation between free GSH, XSSG and G S S G will be designated the glutathione status 2 (table 2). In a similar manner, Kosower 26 described a thiol disulfide status.
Abbreviations: TG = total glutathione, GSH = glutathione, GSSG= disulfide glutathione, XSSG= unsymmetrical glutathione disulfide (as SH-glutathione expressed), NaBH4= sodium borohydride, OPT= ortho-phthalaldehyde, (HPO3) n= recta-phosphoric acid. Supported by DFG (Ha 743/3). 1 F.A. Isherwood, in: Glutathione p.5. Ed. E.M. Crook. Cambridge Univ. Press, Cambridge 1959. 2 G. Harisch and J. Schole, Z. Naturf. C 29, 261 (1974). 3 K.R. Harrap, R.C. Jackson, P.G. Riches, C.A. Smith and B.T. Hill, Biochim. biophys. Acta 310, 104 (1973). 4 S.K. Srivastava, Exp. Eye Res. 22, 577 (1976). 5 H.G. Modig and L. Revesz, Int. J. Rad. Biol. 13, 469 (1967). 6 H.G. Modig, Biochem. Pharmac. 17, 177 (1968). 7 S.K. Srivastava and E. Beutler, Biochem. J. 119, 353 (1970). 8 S.K. Srivastava and E. Beutler, Exp. Eye Res. 17, 33 (1973). 9 B. Mannervik and K. Axelsson, Biochem. J. 149, 785(1975). 10 P. Jocelyn, Eur. J. Biochem. 2, 327 (1967). 11 H.G. Modig, M: Edgren an d L. Revesz, Int. J. Rad. Biol. 22, 257 (1971). 12 P.J. Hissin and R. Hilf, Analyt. Biochem. 74, 214 (1976). 13 H.J. Hohorst, F.H. Kreutz and Th. Bticher, Biochem. Z. 332, 18 (1959). 14 K.P. Faupel, H.J. Seitz, W. Tarnowski, V. Thiemann and Ch. Weiss, Archs Biochem. Biophys. 148, 509 (1972). 15 A.F.S.A. Habeeb, Analyt. Biochem. 56, 60 (1973). 16 E. Beutler and C. West, Analyt. Biochem. 81, 458 (1977). 17 K, Klotzsch and H.W. Bergmeyer, in: Methoden der enzymatischen Analyse, p. 363. Ed. H.U. Bergmeyer. Verlag Chemie, Weinheim 1962. 18 R.J. Jaeger, R.B. Conolly and S.D. Murphy, Res. Commun. chem. Path. Pharmac. 6, 465 (1973). 19 J.J. Harding, Biochem. J. 117, 957 (1970). 20 F. Tietze, Analyt. Biochem. 27, 502 (1969). 21 M. Cernoch, O. Weinbergov~t, V. Manig and P. Vrublovsky, Hoppe Seyler's Z. physiol. Chemic 346, 229 (1966). 22 M. Koivusalo and L. Uotila, Analyt. Biochem. 59, 34 (1974). 23 H. Wernze and W. Koch, Klin. Wschr. 43, 454 (1965). 24 S.K. Srivastva and E. Beutler, Analyt. Biochem, 25, 70 (1968). 25 H. GOntherberg and S. Rapoport, Acta biol. med. germ. 20, 559 (1968). 26 E.M. Kosower, W. Correa, B.J. Kinon and N.S. Kosower, Biochim. biophys. Acta 264, 39 (1972).
Specialia
ciation with this species. Examination at X500 magnification also failed to reveal the presence of obvious contaminant microorganisms. Homogenization of 300 g of freshly collected L birotulata with 600 ml of 0.1 M, pH 7.4 potassium phosphate buffer, followed by centrifugation for 20 rain at 11,000 rpm and recentrifugation of the supernatant for 30 rain at 15,000 rpm (DuPont Sorvall SS-34) gave a dark purple-colored supernatant. The protein precipitate resulting from the 2550% (NH4)2SO 4 fractionation of this supernatant was resuspended in the original volume of phosphate buffer to give the enzyme preparation of interest. Standard assays for peroxidase employing pyrogallol 1~ and dianisidinel~were used to monitor this partial purifcation. The enzyme preparation at this point was only a 2-fold purified product with respect to total protein, but was almost free of the contaminating purple pigment; thus, enabling us to employ spectrophotometric methods in studies of its peroxidase activities. Results and discussion. The oxidation of pyrogallol by the enzyme preparation occurred only in the presence of H202, and was inhibited by both KCN and NaN 3 at 0.01% concentrations. Although the enzyme retained most of its activity upon standing for several days at room temperature, gentle heating destroyed the activity. The initial rate of oxidation and total amount of pyrogallol oxidized within 5 rain was directly related to the amount of enzyme preparation employed (figure). The peroxidase activity was observed throughout the pH range of 4.0-8.0, with 7.5 being the optimal pH. Similar results were obtained with dianisidine as the substrate, although solubility problems prevented pH dependency studies above pH 6.0. The enzyme preparation was unable to effect chlorination of monochlorodimedon a0, nor did it convert 4-chloroaniline to 4-chloronitrosobenzene 6. Thus, we conclude that the peroxidase in L birotulata is similar in properties to horseradish peroxidase rather than chloroperoxidase.
719
Based on pyrogallol oxidation at pH 7.5, the level of peroxidase activity is estimated to be between 12 and 15 ~tM units per g of fresh sponge tissue. This represents an order of magnitude greater concentration of peroxidase in this sponge when compared to a marine red alga known to possess a high level of peroxidase 12. This surprisingly high level of peroxidase activity that we observed in L birotulata makes it highly improbable that this activity could be due to microalgae that might be present on the sponge surface. Our demonstration of the presence of a peroxidase in this sponge necessitates a reconsideration of this biochemical parameter in the Porifera. A survey of sponges for peroxidative enzymes is now underway with the hopes of developing a potential chemotaxonomic tool for the classification of Porifera.
1 This study was supported by grant No.CA 21992 from the National Cancer Institute, DHEW. 2 L. Minale, G. Cimino, S. de Stefano and G. Sodano, Fortschr. Chem. org. NatStoffe 33, 1 (1976). 3 R.J. Wells, Tetrahedron Lett. 2637 (1976). 4 W. Fenical, J. Phycol. 11, 245 (1975). 5 K.H. Wurzinger and R. Hartenstein, Comp. Biochem. Physiol. 49B, 171 (1974). 6 M.D. Corbett, B.R. Chipko and D.G. Baden, Biochem. J. 175, 353 (1978). 7 M.D. Corbett, B.R. Chipko and J.H. Paul, J. envir. Path. Tox. 1, 259 (1978). 8 G.L. Voss, "Seashore Life of Florida and the Caribbean," E.A. Seemann Co., Miami, 1976, p.26. 9 F. Wiedenmayer, Shallow Water Sponges of the Western Bahamas, p. 138. Birkh~user Verlag, Basel 1977. 10 P.F. Hollenberg, T. Rand-Meir and L.P. Hager, J. biol. Chem. 249, 5816 (1974). 11 R. Bartha and L. Bordeleau, Soil Biol. Biochem. 1, 139 (1969). 12 M.J. Murphy and C. Oh Eocha, Phytochemistry 12, 55 (1973).
T h e glutathione status of the rat liver * G. Harisch, J. Eikemeyer and J. Schole
Institute of Physiological Chemistry, College of Veterinary Medicine Hannover, Bisehofsholer Damm 15, D-3000 Hannover (Federal Republic of Germany), 16 October 1978 Summary. F o r the determination of cellular total glutathione, a new method is presented based on a fluorometric procedure. The relation between reduced glutathione, mixed glutathione disulfides and disulfide glutathione will be designated the glutathione status. The tripeptide glutathione is a structural component of most living cells 1. It can be found in 3 forms, as reduced glutathione (GSH), as symmetrical glutathione disulfide (GSSG), and as unsymmetrical glutathione disulfide which we will call XSSG z. In the last form, the glutathione has combined with cysteine-SH groups of cellular proteins ~-9 or with low molecular SH compounds of the cell 1~ to form a disulfide bond. By this means, a 'depot' is developed which, however, in certain situations can very rapidly be mobilized and then is available to the cell as GSH 11. This characteristic makes it appear desirable to determine, in all investigations of cellular thiol-disulfide-status, the percent glutathione bound but mobilizable. Normally this would be done by reducing the disulfide bonds and then measuring the increased G S H level.
However, as there are often only very small quantities available in biological studies, a sensitive and selective GSH assay is necessary. A fluorometric method with these prerequisites was recently presented by Hissin and Hilf 12. We have adapted this method to the conditions in liver tissues and expanded it to include the cellular total glutathione (TG), and thus the portion of bound glutathione can be measured as well. Materials and methods. Male SPF rats, Wistar Han/B6 were kept under defined conditions (24 ~ light from 06.00 till 18.00 h) until they reached the desired 120 g. The animals were anesthetized with 40 mg/kg nembutal i.p. (Pentobarbital Sodium Abbott). The left lobe of the liver was removed using the freeze stop method 13,14. T G measurement: The measurement of cellular total gluta-
720
Specialia
Experientia 35/6
Table 1. Total glutathione (TG) and free glutathione (GSH) GSH added (gg per 80 mg liver) 0 45 TG (gg per 80 mg liver) Expected value Recovery (%) GSH (pg per 80 mg liver) Expected value Recovery (%)
195 + 6.4
139_+5.0
238 _+2.9 240 99 174_+ 1.2 184 95
90
113
135
284-+ 4.4 285 99 226_+ 3.4 229 99
297 -+9.3 308 96 250+_0.4 252 99
326-+ 5.9 330 98 262_+2.4 274 96
Means___SD of 14 liver homogenates from 14 male Wistar rats (120 g b. wt); GSH was added before the homogenation.
thione was done according to the method proposed by Habeeb for splitting smaller amounts of disulfides with NaBH415; however, this method was modified for the conditions necessary for fluorometric G S H measurement: 80 m g fiver powder in 1.75 ml Tris buffer (0.1 M, p H 8.0), in which immediately before the experiment 100 mg NaBH 4 was dissolved, and 2.0 ml guanidine (8 M) were homogenized for 20 sec in an Ultra-Turrax (Jahnke and Kunkel, Staufen, Germany); 0.05 ml n-octanol was used to prevent foaming. The homogenate was incubated (40 ~ for 30 m i n and then cooled in an icebath. 1.0 ml 50% ice cold (HPO3) . was used to deproteinize the sample, which was then centrifuged for 15 rain at 104,000 x g in a vertical rotor (TV 865, D u Pont-Sorvall). 0.5 ml of the supernatant was diluted with 4.5 ml phosphate EDTA-buffer (0.1 M potassium phosphate, 0.005 M EDTA, p H 8.0) T h e n 0.1 ml of the above dilution, 1.8 ml phosphate E D T A buffer and 0.1 ml o-phthalaldehyde (OPT) solution (100 Ixg OPT) were combined and mixed well. After 15 m i n at room temperature, the mixture was transferred to a fluorescence cuvette. The fluorescence intensity was measured in a Shimadzu R F 500 at 423 n m (activation at 350 nm). GSH measurement: The G S H m e a s u r e m e n t corresponded to that of Hissin and Hilf lz. G S H values obtained by the fluorometric method coincide with those determined by the glyoxalase method 1,z. Other low molecular thiols reacting with OPT are quantitativelynegligible 12. G S S G measurement: As the proposed method from Hissin and t-Iilf for measuring fluorometric G S S G 12 is not reliable 16, disulfide glutathione was measured using the enzymatic optic method with glutathione reductase and N A D P H 17. Results and discussion. Glutathione parameters show diurnal 18 and probably also a n n u a l rhythms, and they are dependent on body weight 2. Data o n glutathione in the literature are hardly comparable as they were seldom obtained under identical conditions. 1. Data on T G (table 1) are rather sparse in the literature 3'6. The only exception is the lens of the h u m a n eye 19. Reduced glutathione (GSH) (table 1) is well investigated. Our data are in good agreement with the liver GSH-values obtained with other methods 12'2~ 2. XSSG is calculated by the
Table 2. Glutathione status TG (as GSH)
GSH
gg per g liver 2 4 4 5 _ + 8 0 1741+62 ixmoles per g liver 7.96 5.67 Number of animals 14 14
XSSG ~ (as GSH)
GSSG
636 2.07
67+4 0.110 14
The values are means+_SD; male Wistar rats with 120 g b.wt; see 'results and discussion' section.
formula X S S G = T G - ( G S H + 2 GSSG); XSSG is expressed as SH-glutathione and all data are given in ~tmoles per g liver (table 2). Our results are in coincidence with those of Harrap 3 which were obtained with a different method, and they are in agreement with the results found by Modig with aszites t u m o u r cells 6. 3. The same GSSG-values were obtained by Tietze 2~with a different method. Some authors try to prevent possible G S H oxidation occuring during tissue preparation and they report lower GSSG-values 24,25. 4. The relation between free GSH, XSSG and G S S G will be designated the glutathione status 2 (table 2). In a similar manner, Kosower 26 described a thiol disulfide status.
Abbreviations: TG = total glutathione, GSH = glutathione, GSSG= disulfide glutathione, XSSG= unsymmetrical glutathione disulfide (as SH-glutathione expressed), NaBH4= sodium borohydride, OPT= ortho-phthalaldehyde, (HPO3) n= recta-phosphoric acid. Supported by DFG (Ha 743/3). 1 F.A. Isherwood, in: Glutathione p.5. Ed. E.M. Crook. Cambridge Univ. Press, Cambridge 1959. 2 G. Harisch and J. Schole, Z. Naturf. C 29, 261 (1974). 3 K.R. Harrap, R.C. Jackson, P.G. Riches, C.A. Smith and B.T. Hill, Biochim. biophys. Acta 310, 104 (1973). 4 S.K. Srivastava, Exp. Eye Res. 22, 577 (1976). 5 H.G. Modig and L. Revesz, Int. J. Rad. Biol. 13, 469 (1967). 6 H.G. Modig, Biochem. Pharmac. 17, 177 (1968). 7 S.K. Srivastava and E. Beutler, Biochem. J. 119, 353 (1970). 8 S.K. Srivastava and E. Beutler, Exp. Eye Res. 17, 33 (1973). 9 B. Mannervik and K. Axelsson, Biochem. J. 149, 785(1975). 10 P. Jocelyn, Eur. J. Biochem. 2, 327 (1967). 11 H.G. Modig, M: Edgren an d L. Revesz, Int. J. Rad. Biol. 22, 257 (1971). 12 P.J. Hissin and R. Hilf, Analyt. Biochem. 74, 214 (1976). 13 H.J. Hohorst, F.H. Kreutz and Th. Bticher, Biochem. Z. 332, 18 (1959). 14 K.P. Faupel, H.J. Seitz, W. Tarnowski, V. Thiemann and Ch. Weiss, Archs Biochem. Biophys. 148, 509 (1972). 15 A.F.S.A. Habeeb, Analyt. Biochem. 56, 60 (1973). 16 E. Beutler and C. West, Analyt. Biochem. 81, 458 (1977). 17 K, Klotzsch and H.W. Bergmeyer, in: Methoden der enzymatischen Analyse, p. 363. Ed. H.U. Bergmeyer. Verlag Chemie, Weinheim 1962. 18 R.J. Jaeger, R.B. Conolly and S.D. Murphy, Res. Commun. chem. Path. Pharmac. 6, 465 (1973). 19 J.J. Harding, Biochem. J. 117, 957 (1970). 20 F. Tietze, Analyt. Biochem. 27, 502 (1969). 21 M. Cernoch, O. Weinbergov~t, V. Manig and P. Vrublovsky, Hoppe Seyler's Z. physiol. Chemic 346, 229 (1966). 22 M. Koivusalo and L. Uotila, Analyt. Biochem. 59, 34 (1974). 23 H. Wernze and W. Koch, Klin. Wschr. 43, 454 (1965). 24 S.K. Srivastva and E. Beutler, Analyt. Biochem, 25, 70 (1968). 25 H. GOntherberg and S. Rapoport, Acta biol. med. germ. 20, 559 (1968). 26 E.M. Kosower, W. Correa, B.J. Kinon and N.S. Kosower, Biochim. biophys. Acta 264, 39 (1972).
